Assessment of Newly Synthesized Triazole Compounds Using ZnO(NPs) as Antimicrobial Agents and Theoretical Studies for Inhibiting COVID-19

Abstract An efficient method using ZnO(NPs) was used to synthesize derivatives of s-substituted-1,2,4-triazoles with excellent yield. The structure of all newly formed triazoles was approved using the data extracted from their spectral and elemental analyses. Fifteen s-substituted-triazole derivatives were tested for their antimicrobial activity and the results indicated that triazole derivative 15 recorded the highest antimicrobial activity against Trichophyton mentagrophyte followed by Staphylococcus aureus and Bacillus cereus with clear zone diameters of 30, 28, and 15 mm when compared with the traditional antibiotics, with minimal lethal concentrations of 16, 32, and 64 µg/ml, respectively. While compounds 13c, 13b, 13a and 8a exhibited considerable antimicrobial activity against Gram-positive pathogens including B. cereus, S. aureus. The docking study for the most potent derivatives 8a, 13a, 13b, 13c and 15 against methicillin acyl-Penicillin binding protein 2a from methicillin-resistant S. aureus strain 27r at 2.60 A resolution (PDB: 1MWU) showed strong fit in the active site the methicillin acyl-Penicillin binding protein, especially for derivative 15. Due to the horror caused by the Corona pandemic during the past two years, we docked the s-substituted-1,2,4-triazole derivatives into the active site of SARS-CoV-2 main protease (6LU7). The docking result showed well-fitting and proper orientation of all triazole derivatives except the starting derivative 5. The physicochemical properties, pharmacokinetics, and drug-likeness of the most active s-substituted-1,2,4-triazole derivatives were studied using SwissADME. The results of computational studies and in vitro antimicrobial activity showed great agreement, in addition to the docking study of COVID-19 revealed the ability of triazole derivatives to fit in the active site of the protein, which made us recommend laboratory tests for these triazole derivatives to prove their inhibition efficiency.


Introduction
During the past years, scientists have paid great attention to triazole derivatives due to their effective role as anti-tuberculosis, 1 anti-fungal, 2,3 antioxidant, 4 anti-inflammatory, 5 anti-cancer, 6 antidiabetic, 7 analgesic, 8 anxiolytic 9 and anticonvulsant. 10Dermatophytes are pathogenic fungi that have a strong affinity for the keratinized structures found in the skin, nails, and hair, causing dermatophytosis.The most studied virulence factor is fungal hyphae secretion of a wide range of lytic enzymes, such as proteases and lipases, particularly keratinases, allowing fungi to colonize and maintain in host tissues. 11,12To control onychomycosis, there are currently a reasonable number of antifungal drugs in the market; however, their cellular targets are limited, and fungi may develop tolerance or resistance to these agents.3][14][15][16] The search for new therapeutic options in the treatment of resistant bacterial infections includes the discovery of new synthetic substances as well as natural substances (particularly essential oils extracted from plants. 17,18For the development of pharmaceutically important molecules, a wide range of heterocyclic systems have been explored: Many drugs contain nitrogen-containing heterocyclic compounds, and triazole derivatives have particularly intriguing therapeutic properties. 19Several 1,2,4-triazole derivatives are commercially available as interesting drugs for many diseases as Triazolam, Alprazolam, Etizolam, Propiconazole, Fluconazole, Itraconazole, etc. (Figure 1).The target of many scientists is the synthesis of novel bioactive heterocyclic drugs in short reaction time with high yield.This target can be achieved through using the catalysts, reactions in green solvents or under green heating techniques.Thus, the organic reactions were carried out on the large surface of nanosized-catalysts that accelerate the catalysis process. 20Moreover, the nano-catalysts are easily isolated from the reaction medium with retain their catalytic activity and are recycled. 21The heterogeneous-ZnO nanoparticle can be easily extracted and reused multiple times from the organic reaction medium.Nowadays, ZnO(NPs) are broadly used as an impact for several organic reactions to explore their synthetic advantages, ideally in aqueous solvents or in solvent-free conditions, due to their distinctive new and unique characteristics such as polar surface, reusability, capacity to separate pure compounds, and non-corrosion. 22Nanoparticle catalysts are used in mild conditions to avoid decomposition. 23The Corona pandemic is one of the natural disasters that claimed millions of lives in a short time.This led researchers to find a way to eliminate the virus and limit its spread.][26] Several triazole derivatives showed high docking score that showed binding to the active sites of the 6LU7 protease. 27Continuation of our research program to synthesis bioactive heterocyclic systems, 25,[28][29][30][31][32][33][34] we are interested herein to synthesize a new series of triazole derivatives using ZnO(NPs) and investigate their antimicrobial activity against dermatophytes and the pathogen foodborne bacteria.Due to the urgent needs, all the synthesized s-substituted-1,2,4-triazole derivatives were docked into the active sites of SARS-CoV-2 main protease (6LU7) to detect their ability to inhibit the COVID-19 virous.

Results and discussion
Chemistry 4-Amino-1,2,4-triazole-3-thione derivative was prepared as previously reported 35 as illustrated in Scheme 1 from the reaction of trifluoroacetic acid 1 with thiocarbohydrazide 2 in water under reflux for 5 h.Condensation of the aminotriazolethione ring 3 with 4-nitrobenzaldehyde 4 in acetic acid afforded the respective derivative 5.The structure of the isolated triazole derivative 5 was confirmed by the spectral data as 1 H NMR showed two doublet signals at d ¼ 8.15 and 7.39 ppm for the four protons of aromatic ring and another two singlet signals at d ¼ 8.37 and 10.37 ppm for N¼CH and NH protons, respectively.Novel derivatives of s-substituted-1,2,4-triazole derivatives 8a-e and 9a-d were synthesized in the absence and presence of ZnO(NPs) in dioxane solution from nucleophilic substitution reaction of hydrazonoyl chlorides 6a-e and 7a-d with triazole thione derivative 5 under reflux (Scheme 2).The yields of all derivatives 8 and 9 were found higher in the presence of ZnO nanoparticles as indicated in the experimental part.The excellent yield afforded in the presence of ZnO(NPs) is attributed to the large surface area of nanoparticles of ZnO. 36The structure of all derivatives 8 and 9 were confirmed based on their spectral data (Mass, IR, 1 H NMR, 13 C NMR, and elemental analyses) as for example Figures 2-4.

POLYCYCLIC AROMATIC COMPOUNDS
In detail, the IR spectrum of s-substituted-1,2,4-triazole derivatives 8d revealed the appearance of C¼O absorption band of acetyl group at ¼ 1697 cm À1 in addition to the characteristic absorption band for NH group at 3300 cm À1 (Figure 2).

Antimicrobial activity
Antimicrobial resistance is a growing concern, stubborn disease-causing microorganisms and new pathogens have become serious health problems for humankind worldwide.These conditions encourage pressing desire for the development of a new class of antibiotics, 37 especially a structurally diverse small molecule with a unparalleled mechanism of action from the currently available clinical antimicrobials. 38Recently, triazole compounds have received more interest in drug discovery due to their unparalleled structural properties due to their stability in metabolic decay and capable of hydrogen bonding, which is a preferred side for binding biomolecular targets and can also get better solubility.Because of its polar nature, triazole pulp can raise the solubility of the ligand and can significantly upgrade the biological activity of the drug. 39In this study, fifteen triazole compound were synthesized and the antimicrobial activity were evaluated against Bacillus cereus, and methicillin-resistant Staphylococcus aureus (MRSA) as Gram-positive and Escherichia coli as Gram-negative, Candida albicans and Trichophyton mentagrophyte as dermatophytic fungi (Table 1).The different species of pathogens used in this study were differed in their sensitivity to the triazole compounds, wherein dermatophytic fungi T. mentagrophyte was the most sensitive followed by S. aureus and B. cereus, respectively, while no effect was noticed against either Gramnegative or C. albicans.The variation in antimicrobial activity of tested triazole compounds against pathogen microorganisms may be due to the different thickness of cell wall between Gram-positive and Gram-negative bacteria.Gram-positive and gram-negative bacteria have variations in their membrane structure, the most special of which is the thickness of the peptidoglycan layer. 40Among triazole compounds tested, compound 15 recorded the highest antimicrobial activity against T. mentagrophyte followed by S. aureus and B. cereus with clear zone diameter of Table 1.Antimicrobial activity of newly synthesized Triazoles compounds against dermatophytes and the pathogen foodborne bacteria as indicated by clear zone diameter (CZD, mm) and minimal lethal concentration (MLC, mg/ml).
Clear zone diameter (mm) and minimal lethal concentration (MLC mg/ml) Foodborne pathogenic bacteria Pathogenic molds and fungi B. cereus S. aureus E. coli C. albicans T. mantigrophytes 30, 28 and 15 mm when compared with the traditional antibiotics, with minimal lethal concentration of 16, 32 and 64 mg/ml, respectively (Table 1 and Figure 5).While compound 13c, 13b, 13a and 8a exhibited considerable antimicrobial activity against Gram positive pathogens including B. cereus, S. aureus and weak effect against the dermatophytic fungi of T. mentagrophyte.Similar result was reported by, 16 who found that the inhibitory activity of synthesized triazole compounds against Gram(þ) bacteria was more than that of the gram(-) bacteria, and some triazole derivatives showed moderate or weak activity against C. albicans and Aspergillus niger.Most antibiotics must pass through the outer membrane to reach their targets, for example, hydrophobic drugs can pass through the diffusion pathway, while hydrophilic antibiotics such as -lactams pass through Purine.Vancomycin, on the other hand, cannot cross the outer membrane due to its structure, which prevents it from using any of these passages.Gram-negative bacteria's outer membrane is frequently hidden by a mastic layer, which also conceals the cell's antigens.As a result, it is widely regarded as the primary cause of antibiotic resistance.Gram-negative bacteria can create resistance by altering the outer membrane, such as changing the hydrophobic properties or changing purines and other factors.2][43] In terms of antifungal activity, the previously widely used pharmacological imidazole-based transporter has been replaced by systemically active azoles, owing to lower toxicity and high bioavailability of triazole derivatives, as well as increased specificity of fungal cytochrome p450 and less human effect. 44otably, triazole derivative 15 exhibited strong antimicrobial activity against the dermatophytes including T. mentagrophyte S. aureus; and kept this activity for more than 10 days.Furthermore, no development was recorded when new broth media or agar plates were injected with loops from the pure zone area, referring that compound 15 acting as lethal effect not inhibitor against either Gram-positive pathogen bacteria and/or T. mentagrophyte.Finally, it could be concluded that triazole compounds, particularly triazole compound 15 exhibited antimicrobial activity against the skin infectious dermatophytes such T. mentagrophyte and S. aureus.Therefore, it can serve as an effective anti-fungal alternative for anti-dermatophytes associated with skin infections.Furthermore, the development and design of innovative antimicrobial drugs containing 1,2,4-triazole could aid in the fight against microbial resistance.Moreover, we can comment on the activity of the most potent derivatives 15, 13b and 13a due to the small size and less crowded than derivatives 8 and 9. Also, derivative 15 contains two rich p-electrons phenyl groups that can help in p-H bond with the amino acid of the protein of the bacteria species.

Docking study
Multiple antibiotic impedance of MRSA strains has turn into a main clinical trouble worldwide.Penicillin-binding protein 2a (PBP2a) consider the major determinant of broad-spectrum betalactam resistance in MRSA strains.Due to its low affinity for beta-lactams, PBP2a provides transpeptidase activity to permit cell wall synthesis at beta-lactam concentrations that stop beta-lactam-sensitive PBPs normally generated by S. aureus.The molecular docking study was performed for the promising triazole derivatives 8a, 13a-c and 15 using methicillin acyl-Penicillin binding protein 2a from MRSA strain 27r at 2.60 A resolution (1MWU).The docking results indicated that all s-substituted triazole derivatives showed good interaction with the active site of (1MWU) with docking score ranging from À7.3120 to À6.2425 Kcal/mol.The analysis of the binding profile of 15 demonstrated a high affinity in the 1MWU binding site, with docking scores of À7.312 Kcal/mol.It was observed that the sulfur atom and one of the triazole-nitrogen in all triazole derivatives forming H-bond with the amino acid residue ASP667, GLU351 and ASN555.
The common types of bonds involved in the interaction of the triazoles and the amino acid residues are H-acceptor, H-donor and pi-H (Table 2 and Figure 6).

Docking study for COVID-19 proliferation
The main protease of SARS-CoV-2 is 3CLpro-2, which is a cornerstone in the process of maturation and replication of SARS-CoV-2, which has led to its targeting for the detection of novel drugs for coronavirus. 45The 3CL protease enzyme structure includes three distinct domains (I,  II, and III) made of thirteen strands and nine a-helices.Moreover, in its structure there is a catalytic sabiform consisting of the conserved His41 and Cys145 residues, while the major substrate binding site appears as a division between domains I and II. 46he molecular docking simulation was performed to test the ability of the fifteen s-substituted-triazole derivatives to interact to the active site of COVID-19 main protease (Pdb: 6LU7).The results of this docking study were cited in Table 3 and Figures 7 and 8.The co-crystallized ligand was first redocked for validation.It showed high docking score ¼ À7.7843 kcal/mol that form H-bond acceptor between the C¼O of co-crystallized ligand and SER46 residue (Figure 7).
All investigated s-substituted triazole derivatives showed docking score in the range À5.1911 to À7.9263 kcal/mol.Three triazole derivatives 8d, 11 and 15 revealed docking scores exceeded that for the co-crystallized ligand.These three triazole derivatives 8d, 11 and 15 were fitted in the active site of COVID-19 main protease (Pdb: 6LU7) through H-acceptor, H-donor and pi-H with the amino acid residues THR26, GLN189, HIS41 as illustrated in Figure 8. Sulfur atom and oxygen of nitro group of the s-substituted triazole derivatives were involved in the interaction as listed in Table 3 and Figure 8.These promising results of docking simulation using the COVID-19 major protease (Pdb: 6LU7) lead us to recommend the in vitro investigation of triazole derivatives as antivirals to inhibit the COVID-19 (Figures 8 and 9).

Pharmacokinetics, and ADME activity
The SwissADME website (http://www.swissadme.ch/index.php)has been used to conduct ADMET assessment on all synthetic s-substituted triazole derivatives.SwissADME is a web-based tool that calculates key pharmacokinetic, physicochemical, and drug-likeness variables for substances. 47Tables 4a-c contain data on physicochemical characteristics.From investigation the listed data in Tables 4a-c, we noted that five derivatives (8a,b,e, 13a,b) have molecular weight <500 48 which enhanced the transmissibility of these triazole derivatives to membranes and speed the transport and absorption.The topological polar surface area (TPSA) of triazole derivatives 9a-d, 13a-c and 15 found in the correct published range less than 140 Å2 (Tables 4b and 4c). 50he Lipophilicity (Log P) value of all recorded triazole derivatives are ranged between 3.24-2.27Which agrees with Lipinski's rule of five.Actually, the lipophilicity is related to toxicity and it is demonstrated that the toxicity is significantly higher for derivatives with log Po/w > 5 and TPSA <75 Å2. 51 The obtained results indicate that physicochemical properties of triazole derivatives within the acceptable range.

Conclusion
We constructed a series of substituted triazoles under mild conditions using ZnO(NPs).The structure of all derivatives was confirmed based on their spectral data.All new triazole derivatives were investigated for their antimicrobial activity against dermatophytes and the pathogen foodborne bacteria.The results indicated that five derivatives showed excellent activity with two Foodborne pathogenic bacteria.The docking study for the most potent derivatives 8a, 13a, 13b, 13c and 15 against methicillin acyl-Penicillin binding protein 2a from MRSA strain 27r at 2.60 A resolution (PDB: 1MWU) showed strong fit in the active site the methicillin acyl-Penicillin   derivatives were studied using SwissADME.The results of computational studies and in vitro antimicrobial activity showed great agreement, in addition to the docking study of COVID-19 revealed the ability of triazole derivatives to fit in the active site of the protein, which made us recommend laboratory tests for these triazole derivatives to prove their inhibition efficiency.

Chemistry
All instruments used for spectroscopic measurements are illustrated with pictures with a comprehensive description in the supplementary file.Reaction of 4-[(4-Nitro-benzylidene)-amino]-5-trifluoromethyl-4H-[1,2,4]triazole-3-thiol (5)  with hydrazonoyl chloride derivatives 6a-e, 7a-d, 10, 14 and phenacyl derivatives 12a-c using ZnO(NPs) In round flax, put a mixture of triazolethione derivative 3 (0.002 mole) with each of hydrazonoyl chlorides 6a-e, 7a-d, 10, 14 or phenacyl derivatives 12a-c (0.002 mole) in 20 mL dioxane and ZnO(NPs) 10 mmole and reflux the mixture for 5 h (mentor the reaction progress with TLC.After the reaction was completed, the ZnO(NPs) was separated according to the solubility of the product in certain solvent and remove the solid ZnO(NPs) and the filtrate was evaporated under pressure, then the solid residue was triturated with methanol to give derivatives 8a-e, 9a-d, 11, 13a-c and 15.

Bacterial and fungi strains used in this study
Antimicrobial activity of newly synthesized Triazoles compounds were evaluated against B. cereus DSM 31, and S. aureus (ATCC8095) as Gram-positive pathogenic bacteria, and E. coli (ATCC 25922) as Gram-negative.Also, Triazoles compounds were assessed against the molds and dermatophytes fungi such C. albicans (ATCC 10231) and T. mantigrophytes.Bacterial stock cultures will be kept on nutrient agar slants at 4 C, while fungi and candida yeast will be kept on potato dextrose agar slants at 4 C.

Determination of antimicrobial activity
According to the literature, the antimicrobial activity of fifteen triazole derivatives was evaluated using the agar well diffusion method. 18To accomplish this, sterile Mueller-Hinton agar (for bacteria) and potato dextrose agar (for yeast) were poured into sterile Petri dishes and frozen at room temperature.Overnight bacterial or Candida cultures were removed from agar plates.Using a sterile cork borer, wells with a diameter of 9 mm were created in the center of the agar plates, and 100 mL of each compound triazoles were transferred to the wells.Plates containing pathogenic bacteria were incubated at 37 C for 24 hours, while C. albicans and T. mantigrophytes were incubated at 30 C and 28 C for 24-72 hours, respectively.The antimicrobial activity was determined by measuring the net areas (mm) around each well.As a control, DMSO without the test compounds was used.The antibacterial activity of the antibiotics was determined using the agar disk diffusion method described in 51 by measuring the diameter (mm) of clear areas around each well.Standard antibiotics included fluconazole (100 g/ml), Gentamycin (30 g), Augmentin (30 g), and chloramphenicol (30 g).

Determination of minimum lethal concentrations (MLC)
The minimum lethal concentrations (MLCs) of the newly synthesized triazoles were determined using the dilution method. 18Triazoles (10-500 g/ml) were placed in tubes containing 4 ml of LB or potato dextrose (PD) broth medium, respectively, for pathogenic bacteria or fungi.Each tube will be inoculated with 0.4 mL (0.5 McFarland medium) of a standard suspension of 1 Â 106 CFU/mL of bacterial test species.For pathogenic fungi, two-fold sequential triazole (10-500 mg/ ml) concentrations were injected into tubes containing 4 ml of PD media. 1 Â 106 prepared spores were inoculated into each tube.Each microorganism's inoculated tube was incubated at the suitable temperature and time.Having followed the incubation period, 0.1 ml of each tube has been cultured on LB agar or PDA plates and incubated at the adequate temperature and time for each microorganisms.The least lethal level was calculated to be the lowest of the tested triazoles that generated a viable number of 0.1 percent of the original vaccine (1 Â 106 CFU/mL) (MLC).In antibacterial and antifungal tests, antibiotics for Gram-positive and Gram-negative bacteria, and also fluconazole, will be used as comparison points.

Molecular docking
MOE-Dock 2014 software have been used to record the molecular docking of prepared triazole derivatives. 52Chemical structures of triazole derivatives were drawn in Chem draw program and saved as mol.file followed by opening in MOE and minimized using the program force field MMFF94x.Then, the specific protein type was furnished and the triazole derivatives were prepared and docked by usual way. 30

Figure 5 .
Figure 5. Antimicrobial activity of newly synthesized Triazoles compounds against dermatophytes and the pathogen foodborne bacteria as indicated by clear zone diameter (CZD, mm).

Figure 6 .
Figure 6.2D of docked triazole compounds 8a, 13a-c and 15 into the active site of 1MWU.

Figure 7 .
Figure 7. 2D of docked cocrystal ligand into the active site of 6LU7.

Table 2 .
Docking results of triazole derivatives 8a, 13a-c and 15 with the receptors of (1MWU) for antimicrobial.

Table 3 .
Docking results of s-substituted triazole derivatives with the receptors of (6LU7) for COVID-19.

Table 4a .
Physiochemical and Pharmacokinetics properties for s-substituted triazole derivatives 8a-e.

Table 4b .
Physiochemical and Pharmacokinetics properties for s-substituted triazole derivatives 9a-d and 11.